Exploring the Minimal RNA Substrate of Flexizymes

Abstract

Flexizymes are tRNA acylation ribozymes that have been successfully used to facilitate genetic code reprogramming. They are capable of charging acid substrates onto various tRNAs and tRNA analogues. However, their minimal RNA substrate has not been investigated. Here we have designed fluorescently labeled short RNAs corresponding to the four, three, and two bases (4bRNA, 3bRNA, 2bRNA) at the tRNA 3′‐end and explored the minimal RNA substrate of flexizymes, dFx and eFx. 3bRNA was the observed minimal RNA substrate of the flexizymes, but the efficiency of acylation of this short RNA was two to three times lower than that of 4bRNA. The efficiency of acylation of 4bRNA was comparable with that of the microhelix, a 22‐base RNA conventionally used as a tRNA analogue for analyzing acylation efficiency. We also compared the efficiencies of acylation of the microhelix and 4bRNA with various acid substrates. Thanks to the short length of 4bRNA, its acyl‐4bRNA products exhibited larger mobility shifts in gel electrophoresis than those exhibited by acyl‐microhelix products with every substrate tested. This indicated that 4bRNA was an ideal RNA substrate for analyzing the efficiency of acylation by flexizymes.

Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro

“…Although the malonic acid half esters 19 , 20 , and 21 were poor substrates for the requisite Fx analogue, cyanomethyl ester 22 was a moderate substrate, acylating the acylated MH in 40% yield. Although no gel-shift was observed in the eFx-promoted MH acylation reaction of cyanomethyl ester 23 (perhaps because of low molecular weight and/or polarity), strong evidence for reaction was observed using RNase A/LC-HRMS (Figure C). Indeed, the addition of fMetT derivatives acylated with 22 and 23 (50–100 μM) to PURExpress Δ (aa, tRNA) in vitro translation reactions led to the isolation of polypeptides carrying malonates 22 and 23 ( 22 -VF-FLAG and 23 -VF-FLAG, respectively), whose masses were confirmed by LC-HRMS (Figure D).…”

Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis

“…2A). 25 These showed acylation occurring for both substrates, with the reaction peaking aer 5 hours incubation at 18% for dFx and at 16% for eFx. However, we observed hydrolytic instability during storage with the cyanomethyl ester, and so we opted to continue with the more stable dinitrobenzyl ester for further experiments despite this being atypical for aromatic amino acids.…”

A Label-Free Assay for Aminoacylation of tRNA

“…To test the feasibility of the label-free assay for aminoacylation of tRNA with non-proteinogenic amino acids, we selected several analogs of amino acids that lack a cognate aaRS as a general approach. Because only a limited number of non-proteinogenic amino acids can be charged to tRNA by an aaRS protein enzyme [ 42 ], the general strategy by which to perform aminoacylation is to use the RNA-based dFx flexizyme [ 27 ], which recognizes the universal CCA sequence of tRNA at the 3’-end by base-pairing interaction and catalyzes transfer of the activated form of each amino acid as a dinitro-benzyl ester (DBE) [ 43 , 44 ]. Due to the lack of discrimination of dFx flexizyme among tRNA primary sequences [ 44 ], the tRNA substrate for quantification of aminoacylation should be homogeneous.…”